MICROBIOLOGY PRACTICE BOOKLET

DEGREE IN DENTISTRY
UNIVERSIDAD CATÓLICA DE VALENCIA
Name and Surname:
 MICROBIOLOGY PRACTICAL GUIDE
DEGREE IN DENTISTRY

Practical session 1.

Content:
1. Safety regulations in the laboratory
2. Most used laboratory material
3. Sampling procedures
4. Sample inoculation
5. Antimicrobial sensitivity test

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1. Safety rules in the laboratory

Safety in a microbiology laboratory is important in the prevention of infection

that might be caused by the microorganisms being studied. In addition, many of the
reagents, equipment, and procedures used are potentially hazardous. Attention to
proper procedures and prudent laboratory practices are required for your safety and
protection.

This course does not require the use of any organisms known to be highly
virulent human pathogens. However, some of the organisms used may be potentially
pathogenic. This means that, although they may not cause disease in a normal healthy
human, they might if the body's antimicrobial defence mechanisms are impaired. In
addition, many of the organisms used in this course will be uncharacterized strains
isolated from the environment - these cultures should be handled with care, because
their virulence is unknown.

In addition to organisms, there are some chemicals used in this laboratory

which are potentially harmful. Finally, many procedures involve equipment, glassware,
open flames, and sharp objects which can cause injury if used improperly.

Although none of the organisms, procedures, or materials used in this

laboratory are very dangerous, proper safety techniques and precautions should be
understood and become part of your reflexive laboratory technique. The following
laboratory rules and regulations should be always adhered to, NO EXCEPTIONS.

1. Read and understand each laboratory exercise before you come to class.
2. Follow precautionary statements given in each exercise.
3. The workplace should always be clean and tidy. You should only have the
necessary work material on the laboratory bench. Coats, backpacks, and other
personal belongings will not be allowed on the laboratory bench top. Store
them in a place designated by your instructor or in your laboratory cabinet.
This is to prevent cluttering of the workspace and to avoid exposing them to
permanent stains, caustic chemicals, and microorganisms used in the exercises.
4. Results of every practical work personal or observations should be noted.
5. Disinfect work areas before and after use with 70% ethanol or fresh 10%
bleach. Laboratory equipment and work surfaces should be decontaminated
with an appropriate disinfectant on a routine basis, and especially after spills,
splashes, or other contamination.
6. Spills, cuts and other accidents should be reported to the professor in case
further treatment is necessary.

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7. Do not eat, drink, smoke, or chew pens in the laboratory.

8. Work very close to the Bunsen burner flame to reduce the chances of
contamination (20-30 cm).
9. Turn off Bunsen burners when not is use. Long hair must be restrained if
Bunsen burners are in use.
10. Before using a solution, you must fix it on the label to make sure it is the one
you need and the possible risks of its manipulation. Flammable products (gases,
alcohol, ether, etc.) should be kept away from the flames of the burners.
11. Cover slips and slides should be handled on the edges to prevent surface
contamination with the fingers.
12. Closed toed shoes must be always worn in the lab. Sandals, or flip-flops are not
acceptable. If you want to wear sandals, etc, bring an old pair of
shoes/sneakers and keep them in your drawer.
13. Inoculating loops should be flame sterilized in a Bunsen burner before and
immediately after use to transfer biological material.
14. Wear disposable gloves when working with potentially infectious microbes or
samples (e.g., sewage). If you are working with a sample that may contain a
pathogen, then be extremely careful to use good bacteriological technique.
15. Absolutely no food, drinks, chewing gum, or smoking is allowed in the
laboratory. Keep your fingers and fomites (pencils, etc.) away from your eyes,
nose and mouth. Do not store food in areas where microorganisms are stored.
When handling microorganisms avoid talking or get your hands to your nose,
mouth, eyes etc. Do not touch your eyes or apply makeup in lab, including
Chapstick.
16. Never, under any circumstances, remove equipment, media, or microbial
cultures from the laboratory.
17. Hands must be thoroughly washed before leaving the laboratory.
18. Long hair should be tied back so that it does not catch fire in a Bunsen burner
flame and does not fall into sterile media.
19. Spills should be cleaned immediately. Spills of reagents should be cleaned using
paper towels, followed by a complete rinse with water. If the chemical is
marked 'danger' or 'caustic' you should also notify the prof.
20. Immediately report all accidents such as spills, cuts, burns, or other injuries to
the professor.
21. Know the location of the fire extinguisher and eye wash station.
22. Leave all laboratory facilities and equipment in good order at the end of each
class. Before leaving the laboratory, check to make sure the gas to the Bunsen
burner is turned off.
23. No pets are allowed in the laboratory.

During the semester in the laboratory, you will be taught the methods used in the

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proper handling of microorganisms. Although you will not be working with any that are
human pathogens, exercise caution in handling all material in contact with live
microbial cultures. All cultures should be handled with respect and good technique as
if they were potential pathogens. Specific instructions which should be followed are:
1. Cultures or reagents should not be mouth-pipetted; a pipette bulb or
automatic pipettor should be attached to the pipette.
2. Always keep cultures capped and in proper storage racks or small buckets when
not being used during an exercise.
3. Never place a contaminated pipette, inoculating loop, or any other
contaminated material on the bench top. Flame loops before and after each
use. Place used pipettes in the buckets containing disinfectant. Place all other
contaminated materials in their designated containers. Do not place or put
anything containing live cultures in the sink.
4. Aerosols should be avoided using proper technique for flaming inoculating
loops and by performing any mixing of cultures and reagents in such a way as
to avoid splashing.
5. While agar plates or test tubes are open, all work must be done close to a
Bunsen burner flame where air currents are drawn upwards.
6. On opening a test tube or bottle, the neck must be immediately warmed by
flaming (see below) with the tube held as near to horizontal as possible and so
that any movement of air is outwards from it.
7. If you spill material containing live microorganisms, pour disinfectant on the
spill and notify the laboratory instructor immediately.
8. Cultures of live microorganisms and any material in contact with live cultures
must be properly sterilized after use in the laboratory. Your instructor will
inform you of specific procedures. Follow the general rules outlined below.
a. Glassware such as test tubes, bottles, flasks, and pipettes, is reused and
washed after sterilization. These are normally placed on a cart at the
front of the laboratory after you have finished an experiment or
exercise. Be sure to remove labels before placing any glassware on the
cart. Your instructor will sterilize and then wash these items.
b. Some materials, such as plastic petri dishes, plastic pipettes, microscope
slides, and swabs, are considered disposable. These are used once and if
they become contaminated by contact with live microorganisms are
sterilized and discarded. All these disposable contaminated materials
should be placed in the designated waste container containing an
orange biohazard autoclave bag.

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2. Laboratory materials and equipment

a. Inoculation loops: A tool usually made of platinum wire in which the tip forms
a small loop with a diameter of about 5 mm, and is used to smear, streak, or
take an inoculum from, a culture of microorganisms.

b. Bunsen burner: device to produce a hot clean flame by burning fuel gas in air.
It is common laboratory equipment, which produces a single open gas flame
used for heating, sterilization, and combustion. When air hole is open, air is
drawn into the barrel, where it mixes with the natural gas and ensures
complete combustion. A very hot, blue flame is then produced.

Gas valve

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How to use a Bunsen burner?

- Check for safety: long hair should be tied back, books and papers should be
away from the flame, and the apparatus set up should not be too close to the edge of
the table.
- Check that the air holes are closed: the holes can be adjusted to let in more or
less air by turning the collar.
- Light the Bunsen burner using the match.
- Turn on the gas tap: to turn it on, you must first push down, then turn the tap.
This is a safety feature, so the taps are not accidentally pushed open. Bring the match
to the top of the Bunsen burner.
- Adjust the flame by turning the collar so that you have the appropriate flame
for the experiment.
- Be careful t to avoid accident inside the laboratory.

c. Microscope slides: thin flat piece of glass used to hold objects for examination
under a microscope.

d. Cover slips: Microscope slides are often used together with a cover slip or
cover glass, a smaller and thinner sheet of glass that is placed over the
specimen.

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e. Stains: artificial coloration of a substance to facilitate examination of tissues,

microorganisms, or other cells under the microscope.
• Gram stain: staining procedure in which bacteria are stained with
crystal violet, treated with strong iodine solution, decolorized with
ethanol or ethanol-acetone, and counterstained with a contrasting dye;
those retaining the stain are called gram-positive, and those losing the
stain but staining with the counterstain are called gram-negative.
• Acid-fast stain/Ziehl-Neelsen stain: staining procedure for
demonstrating acid-fast microorganisms.

f. Optical/Light microscope: type of microscope which uses visible light and a

system of lenses to magnify images of small samples.

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• A typical microscope has 3 or 4

objective lenses with different
magnifications, screwed into a circular
“nose piece” which may be rotated to
select the required lens.

• A 4x objective with a 10x eyepiece

produces an image that is 40 times the
size of the object.

Some microscopes use an oil-immersion or water-immersion lens, which can

have magnification greater than 100. These objectives are specially designed for use
with refractive index matching oil or water, which must fill the gap between the front
element and the object. These lenses give greater resolution at high magnification.

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g. Incubators: device used to grow and maintain microbiological cultures or cell

cultures. The incubator maintains optimal temperature, humidity and other
conditions such as the carbon dioxide (CO2) and oxygen content of the
atmosphere inside.

h. Autoclave: pressure chamber used to carry out industrial processes requiring

elevated temperature and pressure different from ambient air pressure.
Autoclaves are used in medical applications to perform sterilization, by
subjecting materials to high-pressure saturated steam at 121 °C (249 °F) for
around 15–20 minutes.

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i. Petri dishes: shallow cylindrical glass or plastic lidded dish that biologists use to
culture cells, such as bacteria.

j. Culture plate: Petri dishes are often used to make agar plates for microbiology
studies. The dish is partially filled with warm liquid containing agar and a
mixture of specific ingredients that may include nutrients, blood, salts,
carbohydrates, dyes, indicators, amino acids or antibiotics. Once the agar cools
and solidifies, the dish is ready to be inoculated ("plated") with a microbe-laden
sample. The most commonly used are blood agar, chocolate agar, McConkey
agar and Muller-Hinton agar.

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k. Pipettes: laboratory tool commonly used in chemistry, biology and medicine to

transport a measured volume of liquid, often as a media dispenser. Pipettes
come in several designs for various purposes with differing levels of accuracy
and precision. They could be made in lastic or glass or can be automatic.

l. Automated equipment: are those equipment that are used in serology for the
detection of antibodies by different methods: enzyme immunoassay ...

m. Thermocycler: laboratory apparatus most commonly used to amplify segments

of DNA via the polymerase chain reaction (PCR).

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n. DNA Sequencer: scientific instrument used to

automate the DNA sequencing process. Given
a sample of DNA, a DNA sequencer is used to
determine the order of the four bases: G
(guanine), C (cytosine), A (adenine) and T
(thymine).

3. Sampling procedures
Sampling is a crucial step for a correct microbiological analysis result. Each
sample requires a special methodology to be known or ask the laboratory prior to
collection. The most frequent samples are those of urine, feces, pharyngeal exudates,
sputum, and respiratory specimens (tracheal aspirates), vaginal and urethral, blood,
wound exudates. The following image shows some of the containers most frequently
used in sampling.

Try to identify each sample with the most correct container.

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a. Collection of throat swab

- Depress the tongue with a tongue depressor.
- Introduce the swab between the tonsillar pillars and behind the uvula without
touching the lateral walls of the buccal cavity.
- Swab back and forth across the posterior pharynx.
- Any exudates or membranes should be taken for specimen.
- Transport within 24hrs to the laboratory.

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4. Bacterial culture:
The objective of inoculating the specimen or a culture of bacteria on to a solid
medium is to obtain discrete colonies of organisms after appropriate incubation.
Hence, a standard technique should be used. Pure cultures can be obtained by the
streak-plate technique. This method is based on the creation of a dilution gradient on
the surface of an agar plate. Due to an appropriate dilution of the inoculum (e.g. mixed
culture), discrete and visible colonies can develop at the end of the inoculation line.
Reisolation of bacterial cells from these colonies (originating from a single cell) onto
agar slants results in pure cultures.

Colony: macroscopically visible collection of millions of bacteria originating from a

single bacterial cell.

After sample collection you will streak the pharyngeal sample on blood agar. To
do this, apply the swab over a lateral sector of the plaque (A), then take the
inoculation loop, sterilize it, and perform the culture as shown in the figure 21.
Incubate the agar plate in the incubator for 24h at 37°C.

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5. Antimicrobial sensitivity test

In clinical microbiology, a microbe is considered sensitive (or susceptible) to an

antimicrobial agent if it is inhibited by a concentration of the drug normally obtained in
human tissues after a standard therapeutic dose. The reverse is true for a resistant
organism. Organisms are considered intermediate in susceptibility if the inhibiting
concentration of the antimicrobial agent is slightly higher than that obtained with a
therapeutic dose.

a. Disc diffusion test:

The disc diffusion test is the most commonly used method of testing the
sensitivity of a microorganism to an antimicrobial agent. Here, the isolate to be tested
is seeded over the entire surface of an agar plate, and drug-impregnated filter paper
discs are applied. After overnight incubation, zones of growth inhibition around discs
indicate sensitivity to the antibiotic, whereas growth of the organism up to the disc
indicates resistance.

Several factors may affect the inhibition growth areas: the antibiotic
concentration on the discs, the diffusion of the antibiotic into the culture medium, the
size of the bacterial inoculum, the composition and thickness of the culture media,
bacterial growth rate and incubation time. The most frequently used culture medium
is Muller-Hinton, which allows growth of almost all bacteria.

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Protocol:
1. Suspend the colonies in saline solution. Special care must be taken in picking
isolated colonies.
2. Starting at the top of the MHA plate inoculate the surface with the
swab. Cover the entire plate by streaking back and forth from edge to edge. Rotate the
plate approximately 60° and repeat the swabbing procedure. Rotate the plate 60°
again and swab the entire plate a third time. This will ensure that the inoculum is
evenly distributed. Let the plate dry for 5 Minutes.
2. Apply the disks containing the antimicrobial agents within 15 minutes of
inoculating the MHA plate. Press each disk down firmly to ensure complete, level
contact with the agar. Don’t forget this step or disks may end up in the lid of the plate
after incubation. Discs should not be less than 15 mm from the edge of the plate and
sufficiently separated between them to that the zones of inhibition do not overlap.
Leave the plates 15 minutes to room temperature.
5. Invert and incubate plates with agar side up at 37°C for 18-24 hours.
6. After the incubation time, antibiotic sensitivity is measured based on the size
of the zone of inhibition (no growth) around each disc.

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Practical session 2.

Content:
1. Bacterial colony morphology
2. Gram staining.
3. Biochemical test for bacterial identification.
4. API 20E strip.

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1. BACTERIAL COLONY MORPHOLOGY

A bacterium (plural bacteria) is a single-celled organism too small to be seen

without a microscope. In order to grow bacteria, known as culturing, the bacteria are
spread onto the surface of agar contained within a petri dish. This agar is gel-like and
contains all the food and nutrients that the bacteria need to grow. As the bacteria
consume the nutrients, they begin to grow and multiply. This generates thousands to
millions to billions of cells that begin to pile up, becoming visible to the naked eye. This
pile of cells originates from one cell and is called a bacterial colony (defined as: “a
visible mass of microorganisms all originating from a single mother cell, therefore a
colony constitutes a clone of bacteria all genetically alike”).

Each species of bacteria produces a colony that looks different from the
colonies produced by other species of bacteria. Examination of the form and structure
of bacterial colonies is termed colony morphology and is one of the first steps in
characterizing and identifying a bacterial culture.

Different types of bacteria will produce different-looking colonies, some

colonies may be colored, some colonies are circular in shape, and others are irregular.
These are the characteristics used to accurately and consistently describe the
morphology of a bacterial colony:

• Size (measure with a millimeter rule): Can vary from large colonies to tiny
colonies less than 1mm = punctiform (pin-point).
• Color (also known as pigmentation): Some bacteria produce pigments, giving
the colony a distinct color. Pigments span the entire color spectrum white, buff,
red, purple, etc. Recording the color is the first step. In addition to describing
the color, this is also the time to identify the colony as opaque (you can't see
through it), translucent (you can see through it), dull, or shiny.
• Texture: refers to the characteristics of the colony surface: butyrous (buttery),
viscid (sticks to loop, hard to get off), brittle/friable (dry, breaks apart), mucoid
(sticky, mucus-like).
• Shape: refers to the overall appearance of the colonies. The descriptors here are
punctiform, circular, irregular, filamentous (has individual thin projections), or
rhizoid (has thin, branching projections).
• Height/elevation: description of how the colony grows vertically. To see the
elevation of the colonies it may be helpful to look through the side of the petri
dish. The descriptors are flat, raised, convex (sloping up from the edges),
pulvinate (sloping steeply from the edges and very high in the center), and
umbonate (has raised center).
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• Edge/margin: describes the borders of the colony. The edge can be entire
(smooth with no projections), undulate (wavy), lobate (lobed) filamentous or
rhizoid.

Colonies of viridans streptococci appear with

a raised center on blood agar plates.

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After the incubation period of 24 hours the plates are visualized to observe bacterial
growth. Describe features of colonies that are displayed and identify the of agar used:

Agar:

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2. GRAM STAINING

This important bacteriological staining procedure was discovered in 1884 by a

Danish scientist, Christian Gram. The staining is based on the cell wall structure of
bacteria. When bacteria are stained with crystal violet, the cells of most Gram-
negative bacteria can be easily decolourised with organic solvents such as ethanol or
acetone, while cells of most Gram-positive bacteria restrict decolourisation. The ability
of bacteria to either retain or lose the stain generally reflects fundamental differences
in the cell wall and is an important taxonomic feature. Gram staining is therefore used
as an initial step in the identification of bacteria.

The bacteria are stained gram positive (+), gram negative (-) or do not stain due
to their differences in the composition of their wall and cellular architecture. Gram
(+) bacteria have a thick layer of peptidoglycan and a large amount of teichoic acids
which are not affected by discoloration with alcohol and/or acetone, retaining the
initial dye complexed with iodine and displaying in varying degrees of tones from violet
to light blue, depending on whether the nature of its cell wall is intact or damaged
(antibiotic treatments, cellular age ...). Gram (-) bacteria have on their cell wall a thin
layer of peptidoglycan bound to an outer membrane by lipopolysaccharide molecules.
This outer membrane is damaged by alcohol and/or acetone from bleaching, allowing
the first dye complexed with iodine to escape and is replaced by the counterstain.

The cells of some bacteria are strongly Gram-positive when young but tend to
become Gram-negative in ageing cultures (e.g. Bacillus cereus, Clostridium spp.), which
may reflect degenerative changes in the cell wall. Some bacteria give a Gram-variable
reaction: they are sometimes Gram-positive, sometimes Gram-negative; this could
reflect minor variation in the staining technique or changes in cell wall thickness, etc.

Gram staining procedure:

1. Preparation of the smear from bacterial plate cultures: with a sterile cooled
loop, place a drop of sterile water or saline solution on the slide. Sterilize and cool the
loop again and pick up a very small sample of a bacterial colony and add to the drop of
water and smear into a very thin layer. Create a thin layer promotes drying as well as
reducing.
2. Heat fixing: kills the bacteria in the smear, firmly adheres the smear to the
slide, and allows the sample to take up stains more readily. Allow the smear to air dry.
After the smear has air-dried, hold the slide at one end and pass the entire slide
through the flame of a Bunsen burner two to three times with the smear-side up.
However, if the slide becomes too hot to the touch, you need to re-start. Now the
smear is ready to be stained.

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Please Note: Take care to prevent overheating the slide because proteins in the
specimen can coagulate causing cellular morphology to appear distorted.

3. Place slide with heat fixed smear on staining tray.

4. Flood the slide with crystal violet stain over the fixed culture and let
stand for 1 minute.
5. Pour off the stain and gently rinse the excess stain with a stream of
water from a faucet or a plastic water bottle, until the water running
out the slide is clear.
6. Add iodine solution on the smear, enough to cover the fixed culture. Let
stand for 1 minute.
7. Pour off the iodine solution and rinse the slide with water. Shake off the
excess water from the surface.
8. Add a few drops of decolourizer (95% ethyl alcohol or acetone), so the
solution trickles down the slide, and let stand for 10-15 seconds.
9. Rinse it off with water after 10-15 seconds. The exact time to stop is
when the solvent is no longer coloured as it flows over the slide. Further
delay will cause excess decolorization in the gram-positive cells, and the
purpose of staining will be defeated.
10. Add fuchsin /safranin to counterstain and let stand for 1 minute.
11. Rinse it off with water.
12. Blot with bibulous paper to remove the excess water.
13. View the smear using a light-microscope under oil-immersion.
14. Place your slide in the specimen holder and begin by viewing your
specimen on the 10x/time objective lens.

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15. The easiest way to focus is to bring your stage as close as possible to the
10x objective lens.
16. Next switch to the 40x objective lens.
17. Move the nosepiece of your microscope so it is between the 40x (high
power objective) and 100x lenses (oil objective) and put a drop of oil on
the slide and swing the 100x immersion lens into the oil. This lens must
touch the oil so that there is a layer of oil between the objective and the
lens.
18. When you finished don’t forget to properly clean and store the
microscope. Remove the slide from the specimen holder and you need
to clean all the objective lenses. Start by cleaning the 10x and 40x lenses
and finish with the 100x lens since it has the oil on it.

1 minute

1 minute

15 seconds

1 minute

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GRAM POSITIVE COCCI

Clusters: usually characteristic of Staphylococcus spp., such as S. aureus.

Chain: usually
characteristic of Streptococcus spp., such as S. pneumoniae, B group streptococci.

Tetrad: usually characteristic of Micrococcus spp.

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GRAM NEGATIVE RODS

Thin rods: usually characteristic of enterobacteriaceae, such as E. coli

o. Optical/Light microscope: type of microscope which uses visible light and a

system of lenses to magnify images of small samples.

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• A typical microscope has 3 or 4

objective lenses with different
magnifications, screwed into a circular
“nose piece” which may be rotated to
select the required lens.

• A 4x objective with a 10x eyepiece

produces an image that is 40 times the
size of the object.

Some microscopes use an oil-immersion or water-immersion lens, which can have

magnification greater than 100. These objectives are specially designed for use
with refractive index matching oil or water, which must fill the gap between the
front element and the object. These lenses give greater resolution at high
magnification.

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3. IDENTIFICATION OF BACTERIA
Bacteria modify the environment around them, capturing substances necessary
for their multiplication and releasing to the environment waste products, enzymes,
exotoxins.... The characterization of these products is a useful tool for bacterial
identification and classification.

• Oxidase test:
Cytochromes are proteins that are part of some electron transport chains,
typical of respirator metabolism. Determining the presence or absence of these
proteins will yield useful information for bacterial classification. The test detects the
presence of cytochrome c in the bacteria. It is based on the ability of the dye
tetramethyl-p-phenylenediamonium of oxidizing by yielding electrons to the
cytochrome c. This colorant is colourless in reduced state and can rapidly oxidize in the
presence of cytochrome giving a blue/purple colour. The oxidase test must be
performed from 5% Sheep blood agar or another medium without a fermentable
sugar.

Procedure of Oxidase test:

1. Take a filter paper soaked with the substrate tetramethyl-p-
phenylenediamine dihydrochloride.
2. Moisten the paper with a sterile distilled water.
3. Pick the colony to be tested with wooden or platinum loop and smear
in the filter paper.
4. Observe inoculated area of paper for a color change to deep blue or
purple within 10-30 seconds.

NEGATIVE

POSITIVE

In general, the cytochrome oxidase system is only found in aerobic organisms,

some facultative anaerobes and, exceptionally, in some microaerophilic, but strict
anaerobes lack oxidase activity.

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• Catalase test:
Catalase is an enzyme, which is produced by microorganisms that live in
oxygenated environments to neutralize toxic forms of oxygen metabolites such as
hydrogen peroxide (H2O2). Hydrogen peroxide is the final product of the oxidative
metabolism of carbohydrates, this metabolic pathway is called aerobic-oxidative
metabolism. The accumulation of peroxide is very toxic so that most facultative
aerobic and anaerobic bacteria except Streptococcus sp. they produce an enzyme
called catalase that degrades hydrogen peroxide (H2O2), obtaining water and oxygen
gas. Thus, the catalase enzyme neutralizes the bactericidal effects of hydrogen
peroxide and protects them. Anaerobes generally lack the catalase enzyme.

Catalase

To find out if a particular bacterial isolate can produce catalase enzyme, small
inoculum of bacterial isolate is mixed into hydrogen peroxide solution (3%) and the
rapid release of oxygen bubbles occurs. The lack of catalase is evident by a lack of or
weak bubble production.

Procedure of catalase test (Slide Test)

1. Transfer a small amount of bacterial colony to a surface of clean, dry glass slide
using a loop or sterile wooden stick.
2. Place a drop of 3% hydrogen peroxide (H2O2) on to the slide and mix.
3. A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced
by bubbling. A negative result is no bubbles or only a few scattered bubbles.
4. Dispose of your slide in the biohazard glass disposal container.

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Catalase-positive bacteria include strict aerobes as well as facultative

anaerobes. They all can respire using oxygen as a terminal electron acceptor.
Catalase-negative bacteria may be anaerobes, or they may be facultative
anaerobes that only ferment and do not respire using oxygen as a terminal electron
acceptor (ie. Streptococcus spp).

The catalase test is primarily used to distinguish among gram-positive cocci:

members of the genus Staphylococcus are catalase-positive, and members of the
genera Streptococcus and Enterococcus are catalase-negative.

Precautions while performing catalase test.

1. Do not use a metal loop or needle with H2O2; it will give a false positive and
degrade the metal.
2. If using colonies from a blood agar plate, be very careful not to scrape up any of
the blood agar as blood cells are catalase positive and any contaminating agar
(carryover of red blood cells) could give a false positive.
3. Because some bacteria possess enzymes other than catalase that can
decompose hydrogen peroxide, a few tiny bubbles forming after 20 to 30
seconds is not considered as positive test.

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4. API (Analytical Profile Index) 20E Test

- API identification products are test kits for identification of Gram positive and
Gram negative bacteria and yeast.
- API strips give accurate identifications based on extensive databases and are
standardized, easy-to-use test systems.
- The kits include strips that contain up to 20 miniature biochemical tests which
are all quick, safe and easy to perform.
- API (Analytical Profile Index) 20E is a biochemical panel for identification and
differentiation of members of the family Enterobacteriaceae.
- It is hence a well-established method for manual microorganism identification
to the species level.

a. Objective
To identify and differentiate members of family Enterobacteriaceae.

b. Principle
The API range provides a standardized, miniaturized version of existing
identification techniques, which up until now were complicated to perform and
difficult to read. In the API 20E, the plastic strip holds twenty mini-test chambers
containing dehydrated media having chemically-defined compositions for each test.
They usually detect enzymatic activity, mostly related to fermentation of carbohydrate
or catabolism of proteins or amino acids by the inoculated organisms.

A bacterial suspension is used to rehydrate each of the wells and the strips are
incubated. During incubation, metabolism produces color changes that are either
spontaneous or revealed by the addition of reagents. All positive and negative test
results are compiled to obtain a profile number, which is then compared with profile
numbers in a commercial codebook (or online) to determine the identification of the
bacterial species.

c. Method
Confirm the culture is of an Enterobacteriaceae. To test this, a quick oxidase
test for cytochrome c oxidase may be performed.
- Pick a single isolated colony (from a pure culture) and make a suspension of it
in sterile distilled water.
- Take the API20E Biochemical Test Strip which contains dehydrated bacterial
media/bio-chemical reagents in 20 separate compartments.
- Using a pasteur pipette, fill up (up to the brim) the compartments with the
bacterial suspension.
- Add sterile oil into the ADH, LDC, ODC, H2S and URE compartments.

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- Put some drops of water in the tray and put the API Test strip and close the
tray.
- Mark the tray with identification number (Patient ID or Organism ID), date and
your initials.
- Incubate the tray at 37oC for 18 to 24 hours.

d. Result Interpretation
For some of the compartments, the color change can be read straightway after
24 hours but for some reagents must be added to them before interpretation.
Add following reagents to these specific compartments:
o TDA: Put one drop of Ferric Chloride
o IND: Put one drop of Kovacs reagent
o VP: Put one drop of 40 % KOH (VP reagent 1) & One drop of VP Reagent
2 (α-Naphthol)

Get the API Reading Scale (color chart) by marking each test as positive or
negative on the lid of the tray. The wells are marked off into triplets by black triangles,
for which scores are allocated.API reading scale
Add up the scores for the positive wells only in each triplet.
Three test reactions are added together at a time to give a 7-digit number,
which can then be looked up in the codebook. The highest score possible for a triplet is
7 (the sum of 1, 2 and 4) and the lowest is 0.
Identify the organism by using API catalog or apiweb (online)

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